In vitro mutagenesis of the v-sis transforming gene defines functional domains of its growth factor-related product.

Abstract

The polypeptide sequence of the v-sis transforming gene product of simian sarcoma virus (SSV) can be divided into four regions that are likely to represent structural domains of the protein. Mutations were generated in the SSV nucleotide sequence to assay the extent or function of each of these regions. The results indicate that the helper virus-derived amino-terminal sequence as well as a core region homologous to polypeptide chain 2 ofplatelet-derived growth factor (PDGF) are required for the transforming function of the protein.Products of transforming but not nontransforming mutants formed dimer structures confornationally analogous to biologically active PDGF.The simian sarcoma virus (SSV) transforming gene, v-sis, is derived from the cellular gene encoding platelet-derived growth factor polypeptide chain 2 (PDGF-2), a major component of PDGF (1-3). Nucleotide sequence analysis of v-sis (4) predicts that only a portion of its gene product is homologous with the PDGF-2 (3-6, 20). While the entire PDGF-2 sequence is encompassed within the v-sis gene product, there are three regions of the predicted protein that are unrelated to PDGF. These include the amino terminus of the transforming protein, which is encoded by sequences derived from the helper virus env gene, as well as two v-sis cell-derived regions immediately flanking the PDGF-2 homologous sequence. These distinct regions of predicted polypeptide sequence suggest a multidomain structure for the v-sis transforming gene product.The processing of the v-sis gene product in cells transformed by SSV also reflects a multidomain structure. The primary v-sis translational product, p28SiS, is much larger than the PDGF-2 polypeptide present in PDGF preparations (7,8). Previous studies have suggested that post-translational modifications of the v-sis gene product result in dimer formation and internal cleavage of amino-and carboxyl-terminal domains to yield a protein analogous to a PDGF-2 dimer (9). In the present study, we have subjected the p283s5 coding sequence to in vitro mutagenesis to determine the contributions of these p28sis domains to the transforming function(s) of the protein. MATERIALS AND METHODSConstruction of SSV Mutations. All SSV variants were derived from a plasmid, pSSVSV2, that contained a biologically active SSV proviral genome previously described (7, 10). The pSV2gpt vector encodes plasmid replication functions and contains a gene that confers resistance to mycophenolic acid and hypoxanthine/aminopterin/thymidine (HAT) media when transfected into eukaryotic cells in culture (11). The structure of each mutant is shown in Fig. 1. pSSVm-l. DNA from pSSVSV2 was isolated from bacteria containing the dam-mutation. As no DNA methylation occurs in this host, plasmid DNA could then be cleaved at the unique Bcl I site located at codon -89. The four-nucleotide overhang of the Bcl I cleavage was blunt-ended with DNA polymerase large fragment, religated with T4 DNA ligase, and used to transform Escherichia coli C600.pSSVm-2. The deletion of cod...

Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation.

“…The connection between dimerization and transformation has recently been described on the basis of two mutants with mutations in the v-sis gene which encode truncated gene products that differ in size by 50 amino acids (17). Our data support previous work with both PDGF and the v-sis gene product, which showed that mitogenic activity was lost upon reduction (20).…”

“…The importance of these Cterminal residues for transformation is not known. A previous report indicated that a truncated 239-amino acid v-sis gene product retains biological activity (17). However, this was only a single endpoint and still retained 19 amino acids beyond the PDGF-homologous region.…”

Immunochemical localization of the epitope for a monoclonal antibody that neutralizes human platelet-derived growth factor mitogenic activity.

“…PDGF BB has been identified as the human homolog of the v-sis oncogene product (4,5,19). The minimal v-sis transforming domain spans 89 amino acids (8,11), contains eight cysteine residues, four of which are involved in intrachain disulfide linkages essential for PDGF function (7,18), and is identical in sequence to PDGF BB. Regions of the molecule specifically involved in PDGF receptor binding and activation have yet to be elucidated.…”

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity.

“…Sequences encoding the remaining 219 amino acids are derived from the woolly monkey PDGF B gene (6). Site-directed mutagenesis has identified the minimum transforming region of v-sis as an 89-codon stretch (12,17). More recent studies have further localized this region to 84 codons bound by cysteine codons (unpublished).…”

“…PDGF BB has been identified as the human homolog of the v-sis oncogene product (7,26,33). The minimal v-sis transforming domain spans 89 amino acids identical in sequence to human PDGF B (12,17) and contains eight cysteine residues, four of which are involved in intrachain disulfide linkages essential for PDGF function (8,31).…”